PROJECT SUMMARY/ABSTRACT The goal is to use novel, yet straightforward DNA vaccines to overcome immune tolerance to tumor associated antigens, thus inducing anti-tumor immunity to provide long-term protection. Her-2 and DR5 death receptors are test vaccine antigens for controlling Her-2 positive and triple negative breast cancer (TNBC), respectively. Novel DNA constructs that combine heterologous and self antigen sequences will be tested as therapeutic cancer vaccines. To amplify vaccine activity, the effect of reducing natural and inducible regulatory T cells and intratumoral expression of immune modulating genes will be tested. To overcome immune tolerance to Her-2, we formulated DNA vaccines that express both human Her-2 and heterologous rat Neu sequences in separate plasmids or as single hybrid constructs that encode Her-2/neu fusion proteins. Candidate vaccines were tested in Her-2 transgenic (Tg) mice of BALB/c (BALB), BALB/c x C57BL/6 F1 (F1) or C57BL/6 (B6) background, which exhibited decreasing immune responsiveness to Her-2. Analysis of various cocktail or hybrid vaccines defined a requirement for particular combination of Her-2/Neu sequences to effectively prime immune effector cells in Her-2 Tg mice. We will test the hypothesis that vaccination with Her-2-neu hybrid DNA vaccine will reject established tumors and prevent tumor metastasis. Proapoptotic death receptor DR5 for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) will be the next vaccine antigen. Human DR5 immune sera induced in wild type mice triggered apoptosis of TNBC and inhibited their growth in vivo. Hybrid human-monkey DR5 vaccines are being constructed to incorporate highly homologous monkey DR5 sequence in human DR5 vaccines. To test vaccine activity in immune tolerant hosts, two human DR5 Tg mouse systems are being generated to mimic immune tolerance in humans. Vaccination with self antigen may trigger the conversion of nave CD4 T cells to induced Treg (iTreg). The role of iTreg in Her-2 vaccine response will be tested in mice deficient of TGF- inducible early gene 1 (TIEG1) and the conversion of their nave CD4 T cells to iTreg is disabled. Further, the effect of TGF- receptor inhibitor (TGF RI) in blocking Treg activity and enhancing Her-2 immune response will be tested. To compliment DNA vaccination with minimal systemic immune modulation, we will exploit the vast antigen reservoir in the solid tumors by intratumoral expression of immune modulating genes. In situ immunization to tumor associated antigens and induction tumor regression will be assessed. Through the testing of these modalities, we aim to define the approach that can most readily be translated into a successful therapeutic vaccine Specifically, we will (1) determine the mechanisms that regulate immune responses to hybrid Her-2 DNA vaccines (2) test the hypothesis that hybrid pE2-NeuTM vaccination combined with complementary immune modulation will reject established tumors, and (3) test the immunogenicity and anti-tumor activity of hDR5 DNA vaccines. The proposed hybrid vaccines complimented with local expression of immune modulating genes and inhibition of Treg, can be expected to achieve complete response with long term immune memory. Importantly, Her-2 and DR5 are expressed by multiple cancer types, thus the impact will reach far beyond breast cancer.